Molecular Analysis of a Salmonella Enterica Group E1 Rfb Gene Cluster: O Antigen and the Genetic Basis of the Major Polymorphism

Salmonella enterica is highly polymorphic for the O antigen, a surface polysaccharide that is subject to intense selection by the host immune system. This polymorphism is used for serotyping Salmonella isolates. The genes encoding O antigen biosynthesis are located in the rfb gene cluster. We report here the cloning and sequence of the 19-kb rfb region from strain M32 (serovar anatum, group E1) and compare it with that of strain LT2 (serovar typhimurium, group B). Genes for biosynthetic pathways common to both strains are conserved and have very similar sequences. In contrast, the five genes for CDP-abequose synthesis, present in strain LT2, are absent in strain M32; three open reading frames (ORFs) of strain LT2, thought to include genes for transferases, are not present in strain M32 but are replaced by three different ORFs with little or low level of similarity. Both rfb gene clusters are low in G + C content, indicating that they were transferred from a common ancestral species with low G + C content to S. enterica relatively recently (in the evolutionary sense). We discuss the recombination and lateral transfer events which may have been involved in the evolution of the polymorphism.     

Cloning and structure of group C1 O antigen (rfb gene cluster) from Salmonella enterica serovar montevideo.

The Salmonella enterica group C1 O antigen structure has a Man-Man-Man-Man-GlcNAc backbone with a glucose branch, which differs from the S. enterica group B O antigen structure which has a Man-Rha-Gal backbone with abequose as side-chain. We have cloned the group C1 rfb (O antigen) gene cluster from serovar montevideo strain M40, using a low-copy-number cosmid vector. The restriction map of the group C1 (M40) rfb gene cluster was compared with that of group B strain LT2 by Southern hybridization and restriction enzyme analysis. The results indicate that the flanking genes are very similar in the two strains, but there is no detectable similarity in the rfb regions. We localized the mannose pathway genes rfbM and rfbK and one of the genes, rfbK, shows considerably similarity to cpsG of strain LT2, suggesting that part of the mannose pathway in the group C1 rfb cluster is derived from a gene of the M antigen (cps) cluster. The M antigen, which forms a capsule, is comprised of four sugars, including fucose. The biosynthetic pathway of GDP-fucose has steps in common with the GDP-mannose pathway, and the cps cluster has isogenes of rfbK and rfbM, presumably as part of a fucose pathway. We discuss the structure and possible evolution of the group C1 rfb gene cluster.     

Cloning of the rfb gene cluster of a group C2 Salmonella strain: comparison with the rfb regions of groups B and D

We report the cloning and mapping of the entire rfb gene cluster of a group C2 Salmonella strain. Comparison with the rfb region of group B strain LT2 and group D strain Ty2 reveals an 11.8 kb central region of limited similarity flanked by regions of high similarity. The genes from the central region confer a group C2 O-antigen structure on a Salmonella LT2 partial delete strain. The significance of this region in relation to function and evolutionary origin is discussed. We also report evidence for the existence of an O-antigen chain-length determinant in Escherichia coli K12 and propose a model for a possible mechanism by which a preferred chain length is determined.     

Relationships among the rfb regions of Salmonella serovars A, B, and D.

The O antigens of Salmonella serogroups A, B, and D differ structurally in their side chain sugar residues. The genes encoding O-antigen biosynthesis are clustered in the rfb operon. The gene rfbJ in strain LT2 (serovar typhimurium, group B) and the genes rfbS and rfbE in strain Ty2 (serovar typhi, group D) account for the known differences in the rfb gene clusters used for determination of group specificity. In this paper, we report the nucleotide sequence of 2.9 kb of DNA from the rfb gene cluster of strain Ty2 and the finding of two open reading frames which have limited similarity with the corresponding open reading frames of strain LT2. These two genes complete the sequence of the rfb region of group D strain Ty2 if we use strain LT2 sequence where restriction site data show it to be extremely similar to the strain Ty2 sequence. The restriction map of the rfb gene cluster in group A strain IMVS1316 (serovar paratyphi) is identical to that of the cluster in strain Ty2 except for a frameshift mutation in rfbE and a triplicated region. The rfb gene clusters of these three strains are compared, and the evolutionary origin of these genes is discussed.     

Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2)

The rfb gene cluster of Salmonella LT2 has been cloned and sequenced. The genes rfbA, rfbB, rfbD, rfbF, rfbG, rfbK, rfbM and rfbP were located individually and the gene rfbL was located outside the cluster. Approximately 16 open reading frames were found in the region which is essential for the expression of O antigen. The gene products of rfbB and rfbG were found to have homology with the group of dehydrogenase and related enzymes described previously. Analysis of the G + C ratio of the rfb cluster extended the area of low-G + C composition previously found in the sequence of rfbJ to the whole rfb gene cluster. Three to five segments with discrete G + C contents and codon adaptation indices are present in the rfb region, indicating a heterogeneous origin of these segments. Potential promoters were found near the start of the rfb region, supporting the possibility that the rfb gene cluster is an operon.     

Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain

The O antigen of Escherichia coli O111 is identical in structure to that of Salmonella enterica serovar adelaide. Another O-antigen structure, similar to that of E. coli O111 and S. enterica serovar adelaide is found in both E. coli O55 and S. enterica serovar greenside. Both O-antigen structures contain colitose, a 3,6 dideoxyhexose found only rarely in the Enterobacteriaceae. The O-antigen structure is determined by genes generally located in the rfb gene cluster. We cloned the rfb gene cluster from an E. coli O111 strain (M92), and the clone expressed O antigen in both E. coli K-12 and a K-12 strain deleted for rfb. Lipopolysaccharide analysis showed that the O antigen produced by strains containing the cloned DNA is polymerized. The chain length of O antigen was affected by a region outside of rfb but linked to it and present on some of the plasmids containing rfb. The rfb region of M92 was analysed and compared, by DNA hybridization, with that of strains with related O antigens. The possible evolution of the rfb genes in these O antigen groups is discussed.     

Biosynthesis of enterobacterial common antigen requires dTDPglucose pyrophosphorylase determined by a Salmonella typhimurium rfb gene and a Salmonella montevideo rfe gene.

In group C1 salmonellae, rfe and rff genes linked to the ilv locus specify the synthesis of a glycolipid called the enterobacterial common antigen. In contrast, in group B salmonellae the synthesis requires in addition some of the genes in the rfb cluster, the main genetic determinant of the O side chains of lipopolysaccharide. In an effort to define the biochemical functions of these rfb genes, we looked in Salmonella typhimurium LT2 (group B) for rfb mutants in which the synthesis of both enterobacterial common antigen and the O side chains would be blocked in a manner suppressible by the wild-type rfe cluster of S. montevideo, of group C1. We found one mutant with these characteristics. This rfb mutation affected the activity of dTDPglucose pyrophosphorylase (glucose-1-phosphate thymidylyltransferase, EC 2.7.7.24). Whereas the rfe cluster of S. montevideo contained a gene producing this enzyme activity, there was no evidence for the presence of such a gene in the rfe cluster of group B strains. These results also showed that the synthesis of dTDP-glucose is necessary for the biosynthesis of enterobacterial common antigen; this conclusion fits with the recent demonstration of 4-acetamido-4,6-dideoxy-D-galactose as a component of enterobacterial common antigen (Lugowski et al., Carbohydr. Res. 118:173-181, 1983), because the biosynthesis of the donor of this sugar, dTDP-4-acetamido-4,6-dideoxy-D-galactose, requires dTDPglucose pyrophosphorylase.     

Complete physical map of the rfb gene cluster encoding biosynthetic enzymes for the O antigen of Salmonella typhimurium LT2.

Biosynthesis of the Salmonella typhimurium LT2 O antigen is encoded by genes which map in the rfb cluster. The cloning and restriction enzyme analysis of part of this cluster have been described previously (H. N. Brahmbhatt, N. B. Quigley, and P. R. Reeves, Mol. Gen. Genet. 203:172-176, 1986). The entire rfb gene cluster has now been cloned, and a detailed restriction enzyme map has been constructed which has enabled us to map the approximate positions of individual rfb genes.     

Molecular analysis of the rfb O antigen gene cluster of Salmonella enterica serogroup O:6,14 and development of a serogroup-specific PCR assay

The Kauffmann-White scheme for serotyping Salmonella recognizes 46 somatic (O) antigen groups, which together with detection of the flagellar (H) antigens form the basis for serotype identification. Although serotyping has become an invaluable typing method for epidemiological investigations of Salmonella, it does have some practical limitations. We have been characterizing the genes required for O and H antigen biosynthesis with the goal of developing a DNA-based system for the determination of serotype in Salmonella. The majority of the enzymes involved in O antigen biosynthesis are encoded by the rfb gene cluster. We report the sequencing of the rfb region from S. enterica serotype Sundsvall (serogroup O:6,14). The S. enterica serotype Sundsvall rfb region is 8.4 kb in length and comprises six open reading frames. When compared with other previously characterized rfb regions, the serogroup O:6,14 sequence is most related to serogroup C(1). On the basis of DNA sequence similarity, we identified two genes from the mannose biosynthetic pathway, two mannosyl transferase genes, the O unit flippase gene and, possibly, the O antigen polymerase. The whole cluster is derived from a low-G+C-content organism. Comparative sequencing of an additional serogroup O:6,14 isolate (S. enterica serotype Carrau) revealed a highly homologous sequence, suggesting that O antigen factors O:24 and O:25 (additional O factors associated with serogroup O:6,14) are encoded outside the rfb gene cluster. We developed a serogroup O:6,14-specific PCR assay based on a region of the putative wzx (O antigen flippase) gene. This provides the basis for a sensitive and specific test for the rapid identification of Salmonella serogroup O:6,14.     

Sequence and structural analysis of the rfb (O antigen) gene cluster from a group C1 Salmonella enterica strain.

The rfb (O antigen) gene cluster of a group C1 Salmonella enterica strain was sequenced; it comprised seven open reading frames which precisely replaced the 16 open reading frames of a group B strain. Two genes of the mannose biosynthetic pathway were present: rfbK (phosphomannomutase) had a G+C content of 0.61 and had only 40% identity to rfbK of group B but was very similar to cpsG of the capsular polysaccharide pathway with 96% identity, whereas rfbM [guanosine diphosphomannose (GDP-Man) pyrophosphorylase] had a G+C content of 0.39. Other genes had G+C contents ranging from 0.24 to 0.28. rfbM(C1) and rfbM(B) had 60% identity, which is much less than expected within a species, but nonetheless indicates a much more recent common ancestor than for rfbK. The other genes showed much lower or no similarity to rfb genes of other S. enterica strains. It appears that the gene cluster evolved outside of Salmonella in a species with low G+C content: the rfbM gene presumably derives from that period whereas the rfbK gene appears to have arisen after transfer of the cluster to S. enterica by duplication of the S. enterica cpsG gene, presumably replacing an rfbK gene of low G+C content.     

Two functional O-polysaccharide polymerase wzy (rfc) genes are present in the rfb gene cluster of Group E1 Salmonella enterica serovar Anatum

Defined regions of the rfb gene cluster of Group E1 Salmonella enterica serovar Anatum were introduced into a mutated derivative of this strain that lacks O-polysaccharide polymerase activity. Three different kinds of assays performed on the various transformants all indicate that two functional wzy (rfc) genes reside within the Group E1 Salmonella rfb gene cluster. The product of ORF9.6, positioned near the center of the rfb gene cluster, joins O-polysaccharide repeat units together by alpha-glycosidic linkages to produce antigen O10, the major serological determinant of Group E1 S. enterica. The product of ORF17.4, positioned at the downstream end of the rfb gene cluster, can join repeat units together by beta-glycosidic linkages to produce antigen O15, the major serological determinant of Group E2 S. enterica.     

Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster.

Escherichia coli K-12 has long been known not to produce an O antigen. We recently identified two independent mutations in different lineages of K-12 which had led to loss of O antigen synthesis (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994) and constructed a strain with all rfb (O antigen) genes intact which synthesized a variant of O antigen O16, giving cross-reaction with anti-O17 antibody. We determined the structure of this O antigen to be -->2)-beta-D-Galf-(1-->6)-alpha-D-Glcp- (1-->3)-alpha-L-Rhap-(1-->3)-alpha-D-GlcpNAc-(1-->, with an O-acetyl group on C-2 of the rhamnose and a side chain alpha-D-Glcp on C-6 of GlcNAc. O antigen synthesis is rfe dependent, and D-GlcpNAc is the first sugar of the biological repeat unit. We sequenced the rfb (O antigen) gene cluster and found 11 open reading frames. Four rhamnose pathway genes are identified by similarity to those of other strains, the rhamnose transferase gene is identified by assay of its product, and the identities of other genes are predicted with various degrees of confidence. We interpret earlier observations on interaction between the rfb region of Escherichia coli K-12 and those of E. coli O4 and E. coli Flexneri. All K-12 rfb genes were of low G+C content for E. coli. The rhamnose pathway genes were similar in sequence to those of (Shigella) Dysenteriae 1 and Flexneri, but the other genes showed distant or no similarity. We suggest that the K-12 gene cluster is a member of a family of rfb gene clusters, including those of Dysenteriae 1 and Flexneri, which evolved outside E. coli and was acquired by lateral gene transfer.     

Molecular analysis of the rfb gene cluster of a group D2 Salmonella enterica strain: evidence for its origin from an insertion sequence-mediated recombination event between group E and D1 strains.

The Salmonella enterica O antigen is a highly variable surface polysaccharide composed of a repeated oligosaccharide (the O unit). The O unit produced by serogroup D2 has structural features in common with those of groups D1 and E1, and hybridization studies had previously suggested that the D2 rfb gene cluster responsible for O-unit biosynthesis is indeed a hybrid of the two. In this study, the rfb gene cluster was cloned from a group D2 strain of S. enterica sv. Strasbourg. Mapping, hybridization, and DNA sequencing showed that the organization of the D2 rfb genes is similar to that of group D1, with the alpha-mannosyl transferase gene rfbU replaced by rfbO, the E1-specific beta-mannosyl transferase gene. The E1-specific polymerase gene (rfc) has also been acquired. Interestingly, the D1-like and E1-like rfb regions are separated by an additional sequence closely related to an element (Hinc repeat [H-rpt]) associated with the Rhs loci of Escherichia coli. The H-rpt resembles an insertion sequence and possibly mediated the intraspecific recombination events which produced the group D2 rfb gene organization.     

Molecular analysis of the 3,6-dideoxyhexose pathway genes of Yersinia pseudotuberculosis serogroup IIA.

Salmonella enterica and Yersinia pseudotuberculosis are the only examples in nature known to use a variety of 3,6-dideoxyhexose derivatives as O antigen constituents. To allow a comparison of the responsible biosynthetic genes of the two organisms, we have sequenced a section of the Y. pseudotuberculosis serogroup IIA rfb region that contained the genes for the abequose biosynthetic pathway. Comparison of the identified genes with the rfb region of S. enterica LT2 showed that the two dideoxyhexose pathway gene clusters are related. The arrangement of the genes was largely conserved, and the G + C compositions of the two DNA regions were strikingly similar; however, the degree of conservation of nucleotide and protein sequences suggested that the two gene clusters have been evolving independently for considerable time. Hybridization experiments showed that the dideoxyhexose pathway genes are widespread throughout the various serogroups of Y. pseudotuberculosis.     

Glycosyl transferases of O-antigen biosynthesis in Salmonella enterica: identification and characterization of transferase genes of groups B, C2, and E1.

In Salmonella enterica, there is a great variety of O antigens, each consisting of a short oligosaccharide (the repeating unit) repeated many times. The O antigens differ in their sugar composition and glycosidic linkages. The genetic determinants of the O antigen are located in an rfb gene cluster, and some, including those of S. enterica O serogroups B, C2, and E1, have been cloned and sequenced. In this study of the glycosyltransferases which form the glycosidic linkages, we identify and characterize the four mannosyl and three rhamnosyl transferase genes of the three rfb gene clusters.     

Presence of rfe genes in Escherichia coli: their participation in biosynthesis of O antigen and enterobacterial common antigen.

In Salmonella, ilv-linked rfe genes participate in the biosynthesis of the enterobacterial common antigen (CA) as well as of certain types of O antigen (serogroups C1 and L). rff genes, probably in the same cluster with rfe, are required for CA synthesis (P.H. M?kel? et al., in preparation). Several Escherichia coli strains were studied to determine whether they also have rfe-rff genes that are involved in the synthesis of O antigen and CA, or of CA only. In a first approach, E, coli K-12 F-prime factors carrying the genes ilv and argH or argE and presumably rfe-rff genes were introduced into CA-negative Salmonella mutants that are blocked in CA synthesis because of mutated rfe or rff genes. All resulting ilv+ hybrids were CA positive. In recipients with group C1-derived rfb genes, the synthesis of O6,7-specific antigen was also restored. This result shows that E. coli K-12 has rfe and rff genes providing the functions required in the synthesis of CA and Salmonella 6,7-specific polysaccharide. By introduction of defective rfe regions from suitable Salmonella donors into E. coli O8, 09, and O100 strains, the synthesis of CA as well as of the O-specific polysaccharides was blocked. This indicates that in the E. coli strains tested the rfe genes are involved in the synthesis of both O antigen and CA. This suggestion was confirmed by the finding of E. coli rough mutants that had simultaneously become CA negative. In transduction experiments it could be shown that the appearance of the rough and CA- phenotype was due to a defect in the ilv-linked rfe region.     

Construction of Salmonella strains with both antigen O4 (of group B) and antigen O9 (of group D).

A Salmonella live vaccine causing both O4- and O9-specific immune responses would be of use, but no reported Salmonella serotype has both of these O antigens. Constructed Salmonella typhimurium strains with an rfb (O-antigen-specifying) gene cluster of type D in the chromosome and one of type B in an F'-rfb+ factor, and those with the reverse combination reacted strongly with both anti-O4 (and anti-O5) and anti-O9 sera and, if they carried recA, could be maintained in this state by growth conditions selective for retention of the F' factor. One of the two B.rfb+ gene clusters of a (P22-lysogenic) S. typhimurium strain with a tandem duplication of a chromosomal segment including hisD and B.rfb+ was replaced (by transduction) by a D.rfb+ gene cluster; the resulting strain was O1+ O4+ O5+ O9+ and stable as such after being made recA. A stable O4+ O9+ derivative of a virulent S. enteritidis (O-group D) strain was made by transducing into it first the join point of an appropriate tandem duplication strain, together with the adjacent B.rfb+ gene cluster, and then srl::Tn10 recA.     

Transferases of O-antigen biosynthesis in Salmonella enterica: dideoxyhexosyltransferases of groups B and C2 and acetyltransferase of group C2.

The O antigen is a polymer of oligosaccharide units. O antigens differ in their sugar composition and glycosidic linkages, and genes responsible for O-antigen-specific biosynthesis are grouped in the rfb gene cluster. In this study, we identified two abequosyltransferase genes and an acetyltransferase gene in Salmonella enterica groups B and C2 by in vitro assay and identified paratosyl-, tyvelosyl-, and abequosyltransferase genes from S. enterica groups A and D and Yersinia pseudotuberculosis serovar IIA, respectively, by comparison.     

Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110: identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a.

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.     

Sequence analysis of the rfb loci, encoding proteins involved in the biosynthesis of the Salmonella enterica O17 and O18 antigens: serogroup-specific identification by PCR

We report sequencing of the O antigen encoded by the rfb gene cluster of Salmonella enterica serotype Jangwani (O17) and Salmonella serotype Cerro (O18). We developed serogroup O17- and O18-specific PCR assays based on rfb gene targets and found them to be sensitive and specific for rapid identification of Salmonella serogroups O17 and O18.